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ORIGINAL RESEARCH ARTICLE

Engineering Redox-Sensitive Linkers for Genetically Encoded FRET-Based Biosensors

Vladimir L. Kolossov^*,,1, Bryan Q. Spring^,#, Anna Sokolowski^*,, John E. Conour^,2, Robert M. Clegg^*,,#, Paul J. A. Kenis^*, and H. Rex Gaskins^*,,||,¶

^* Institute for Genomic Biology, Departments of Animal Sciences, Physics, Chemical & Biomolecular Engineering, ^|| Pathobiology, ^¶ Division of Nutritional Sciences, and ^# Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

To whom requests for reprints should be addressed at ¹ 1206 W. Gregory Dr. Urbana, IL 61801. E-mail: viadimer{at}uiuc.edu or hgaskins{at}uiuc.edu

The ability to sense intracellular or intraorganellar reduction/oxidation conditions would provide a powerful tool for studying normal cell proliferation, differentiation, and apoptosis. Genetically encoded biosensors enable monitoring of the intracellular redox environment. We report the development of chimeric polypeptides useful as redox-sensitive linkers in conjunction with Förster resonance energy transfer (FRET). -helical linkers differing in length were combined with motifs that are sensitive to the redox state of the environment. The first category of linkers included a redox motif found in the thioredoxin family of oxidoreductases. This motif was flanked by two -helices of equal length. The second and third categories of redox linkers were composed of -helices with embedded adjacent and dispersed vicinal cysteine residues, respectively. The linkers containing redox switches were placed between a FRET pair of enhanced cyan and yellow fluorescent proteins and these constructs were tested subsequently for their efficacy. A robust method of FRET analysis, the (ratio)_A method, was used. This method uses two fluorescence spectra performed directly on the FRET construct without physical separation of the fluorophores. The cyan/yellow construct carrying one of the designed redox linkers, RL5, exhibited a 92% increase in FRET efficiency from its reduced to oxidized states. Responsiveness of the cyan-RL5-yellow construct to changes in the intracellular redox environment was confirmed in mammalian cells by flow cytometry.

Key Words: redox-sensitive switch • alpha-helical linker • green fluorescent protein (GFP) variants • genetically encoded biosensor • Förster resonance energy transfer (FRET) • FRET efficiency measurements

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Engineering Chimeric Polypeptides to Illuminate Cellular Redox States

EBM 2008 233: vii. [Full Text]